Nucleoside reverse transcriptase inhibitors (NRTI) are critical components of highly active anti-viral therapies. Phosphorylated derivatives (NRTI-P) mimic the cellular dNTP substrates for HIV RT, but produce chain termination, thereby limiting retroviral amplification. NRTIs produce reversible side effects that resemble those of patients with mitochondrial (mito) genetic disorders, including cardiomyopathy, myopathy, neuropathy and lactic acidosis, and these correlate with NRTI-induced loss of mito DNA in various tissues. Toxicity has been attributed to inhibition of DNA polymerase g, interfering with mito DNA maintenance. Alternative or additional mechanisms of mito damage may also be at play. Regardless of mechanism, NRTI toxicity is dependent on import into mito. We have isolated novel human genes and cDNAs that encode orphan members of the mito metabolite carrier family (OMC). Five carry a signature specific to nucleotide (nt) carriers, including a putative adenine nt transporter (ANT), two putative CoA transporters, and a non-specific exchanger of nucleotides (OMC27). We have shown that purified OMC27 and its yeast ortholog transport NRTI-P's AZT-TP and -DP, ddl-TP, and ddC-TP in proteoliposome radiochemical flux assays. Disruption of the orthologous yeast gene protects against AZT-induced mito dysfunction. This project examines the role of OMC27 in NRTI-mediated toxicity, with goals of understanding mechanisms by which NRTIs gain entry to the matrix, and identifying nucleoside analogs with known anti-viral activity that offer potential for improved therapeutic index by virture of mito exclusion and reduced mito toxicity. We will test the hypotheses that OMC27 (and yeast ortholog) are highly affinity transporters of NRTI-P, that this represents the primary pathway for mito uptake of NRTI-P; that OMC27 expression is critical to NRTI-induced mito failure; and that a useful therapeutic index for NRTIs may be evident from the ratio of NRTI anti-viral to MC-mediated uptake activities; using complementary in vitro, yeast and mammalian cell, and in vivo mouse studies.